CN107158299A - 一种具有增强学习记忆力功能的组合物及其制备方法 - Google Patents
一种具有增强学习记忆力功能的组合物及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种具有增强学习记忆力功能的组合物及其制备方法,该组合物主要有效成分由以下重量份的原料制备而成:蚕蛹10‑30份,石菖蒲2‑10份,远志2‑10份,人参1‑5份,五味子1‑5份,益智仁1‑5份,肉苁蓉1‑5份,菟丝子1‑5份。该组合物能有效增强学习记忆力作用,同时还是抗衰老抗氧化和增强免疫力的代表保健品,尤其适用于老年学习记忆力减退患者。
Description
技术领域
本发明涉及组合物,具体是增强学习记忆力功能的组合物,属于保健品领域。
背景技术
现代社会工作、学习节奏不断加快,社会压力增大,导致许多人出现了不同程度的学习记忆力衰退情况,严重影响了正常生活。如今,学习记忆力减退已成为临床热点问题之一,开发改善或治疗这类疾病的保健品及药物已刻不容缓。目前,临床上针对该症状,主要采用镇静催眠及调节神经系统药物进行辅助干预,效果不佳;另外目前多采用化学药物,毒副作用较大,不适于长期使用。
中医中药在我国有着悠久的历史,有“药食同源”的理论作基础,来源于天然产物,毒副作用较小,可以长期服用,因此特别适合于学习记忆力减退等亚健康状态的调理和治疗。因此,从天然中药及食材中寻找具有改善学习记忆力的活性成分,并据此开发药物和保健食品已成为研究热点,具有十分重要的现实意义。
发明内容
基于此,本发明的目的在于克服现有技术的缺陷,提供一种用于增强学习记忆力的组合物,该组合物具有很好的增强学习记忆力的作用。为实现上述目的,本发明采取以下技术方案:
一种用于增强学习记忆力的组合物,所述组合物的有效成分由以下重量份的原料制备而成:
蚕蛹10-30份,石菖蒲2-10份,远志2-10份,人参1-5份,五味子1-5份,益智仁1-5份,肉苁蓉1-5份,菟丝子1-5份。
另外的,一种用于增强学习记忆力的组合物的有效成分由以下重量份的原料制备而成:
蚕蛹10-15份,石菖蒲2-3份,远志2-3份,人参1-1.2份,五味子1-1.2份,益智仁1-1.2份,肉苁蓉1-1.2份,菟丝子1-1.2份。
另外的,一种用于增强学习记忆力的组合物的有效成分由以下重量份的原料制备而成:
蚕蛹10份,石菖蒲2份,远志2份,人参1份,五味子1份,益智仁1份,肉苁蓉1份,菟丝子1份。
本发明还公开一种上述的用于增强学习记忆力的组合物的制备方法,包括以下步骤:
制备浸膏一:按重量份称取蚕蛹,粉碎,得粗粉,按蚕蛹原料重量的10-15倍加水浸泡,随后以盐酸调节溶液pH值为1.5-2.5,再按蚕蛹重量百分比的0.3-0.6%加入胃蛋白酶和按蚕蛹重量百分比的0.3-0.6%加入胰蛋白酶,37℃酶解3-6小时,然后加热使酶失活,过滤,得酶解液,将酶解液浓缩,即得浸膏一;
制备浸膏二:按重量份称取石菖蒲、远志和人参,按石菖蒲、远志和人参总重量的6-10倍加入体积百分含量为60%-80%乙醇溶液,回流提取2-4次,每次回流提取时间为2-4小时,合并滤液,将滤液浓缩,即得浸膏二;
制备浸膏三:按重量份称取五味子、益智仁、肉苁蓉和菟丝子,按五味子、益智仁、肉苁蓉和菟丝子总重量的10-15倍加入水,在80-100℃下浸提2-4次,每次浸提时间为2-4小时,合并滤液,将滤液浓缩,即得浸膏三;
混合:将上述得到的浸膏一、浸膏二和浸膏三混合均匀,即得具有增强学习记忆力功能的组合物。该组合物再经真空干燥,粉碎,得到粉末状的浸膏,命名为组合物1。
本发明的一种用于增强学习记忆力的组合物及其制备方法,采用复合生物酶解超滤技术提取蚕蛹有效成分,工艺方法独特、条件温和,可在有效提取蚕蛹活性成分的同时保留其生物活性,使得蚕蛹有效成分得到最大限度的利用。且该制备方法中,工艺科学合理,质量稳定、可控、便于操作,可实现工业化连续生产,生产成本低。制备的组合物具有良好的增强学习记忆力作用,同时还是抗衰老抗氧化和增强免疫力的代表中药,尤其适用于老年学习记忆力减退患者。并且,这些原料均为食材或药食两用的药材,符合中医“药食同源”理论,天然、毒副作用低,适合长期服用。
具体实施方式
下面详细描述本发明的具体实施方式,本发明不受下述实施案例限制,可根据本发明的技术方案与实际情况确定具体的实施方式。在描述中,在此所使用的某些术语仅用于参照,并不意味着进行限制。
本发明采取以下技术方案:
一种用于增强学习记忆力的组合物,所述组合物的有效成分由以下重量份的原料制备而成:
蚕蛹10-30份,石菖蒲2-10份,远志2-10份,人参1-5份,五味子1-5份,益智仁1-5份,肉苁蓉1-5份,菟丝子1-5份。
方中:蚕蛹为蚕蛾科昆虫家蚕Bymbyx mori (Linnaeus) 的干燥蛹。味甘、辛、咸,性微温。归脾、胃、肝、肾经。蚕蛹《本草纲目》、《日华子本草》、《东医宝鉴》、《医林纂要》、《圣惠方》、《圣济总录》等古代医药经典著作中均有详细记载,蚕蛹具有生津止渴、消食理气、镇惊、壮阳、和脾胃、祛风湿、长肌退热等功效,用于治疗虚损、小儿疳瘦、心血管疾病、慢性肝炎、肾阳不足等疾病,此外,还有滋补强壮作用。现代研究表明,蚕蛹中富含蛋白质、脂肪、维生素A、B2、D和麦角甾醇等物质。具有增强学习记忆力、降血糖、降血脂、抑制血小板凝集等功效。在增强学习记忆力方面,蚕蛹能明显地提高正常小鼠跳台被动回避反应再现能力和提高避暗反应再现能力,具有明显改善记忆作用。还能显著提高D-半乳糖所致衰老小鼠脑组织SOD、GSH-PX活性,降低脂质过氧化产物MDA的含量,具有改善D-半乳糖致衰老小鼠的学习记忆能力的作用。
石菖蒲为天南星科植物石菖蒲Acorus tatarinowii Schott 的干燥根茎。味辛、苦,性温。归心、胃经。具有化湿开胃,开窍豁痰,醒神益智的功效。用于脘痞不饥,噤口下痢,神昏癫痫,健忘耳聋等症。现代研究表明,石菖蒲中含有挥发油、糖类、有机酸、氨基酸等成分。具有调节中枢神经系统、植物神经系统、内分泌系统、心血管系统和消化系统的作用,其最主要的药理作用表现为中枢镇静抗惊作用。此外,药理实验证明,与石菖蒲配伍的复方制剂能对抗樟柳碱引起的小鼠记忆障碍,提高正常小鼠的学习记忆能力,增加大鼠脑内去甲肾上腺素(NA)和多巴胺(DA)的含量,减少5-羟色胺(5-HT)、5-羟吲哚乙酸(5-HIAA)含量及增加脑内蛋白质和核酸的含量。
远志为远志科植物远志Polygala tenuifolia Willd.的干燥根。味苦、辛,性微温。归心、肾、肺经。具有安神益智,祛痰,消肿的功效。用于心肾不交引起的失眠多梦,健忘惊悸,神志恍惚,咳痰不爽,疮疡肿毒,乳房肿痛等症。现代研究表明,远志中含有多种皂苷、口山酮、糖酯、生物碱类物质。具有增强学习记忆力、镇静、中枢降压、抗氧化、抗衰老等作用。
人参五加科植物人参Panax ginseng C. A. Mey.的干燥根。味甘、微苦,性平。归脾、肺、心经。具有大补元气,复脉固脱,补脾益肺,生津,安神等功效。用于体虚欲脱,肢冷脉微,脾虚食少,肺虚喘咳,津伤口渴,内热消渴,久病虚羸,惊悸失眠,阳痿宫冷;心力衰竭,心原性休克等症。现代研究表明,人参中含皂甙、挥发油、多糖、有机酸、黄酮类物质和多种维生素。具有兴奋神经系统、调节血压和血糖、抗氧化、抗衰老、提高视力等作用。
五味子为木兰科植物五味子Schisandra chinensis (Turcz.)Baill.或华中五味子Schisandra sphenanthera Rehd. et Wils.的干燥成熟果实。味酸、甘,性温。归肺,心、肾经。具有收敛固涩,益气生津,补肾宁心之功效。用于久嗽虚喘,梦遗滑精,遗尿尿频,久泻不止,自汗,盗汗,津伤口渴,短气脉虚,内热消渴,心悸失眠等症。现代研究表明,五味子中主要含有多种木脂素类物质、多糖和挥发油。具有调节中枢神经系统、提高学习记忆力、促进肝糖原生成、提高免疫力等作用。
益智仁为姜科植物益智Alpinia oxyphylla Miq.的果实。味辛,性温。归脾、肾经。具有温脾止泻摄涎,暖肾缩尿固精的功效。用于脾胃虚寒,呕吐,泄泻,腹中冷痛,口多唾涎,肾虚遗尿,尿频,遗精,白浊等症。含有挥发油、并含丰富的B族维生素及维生素C,以及微量元素锰、锌、钾、钠、钙、镁、磷、铁、铜等。具有调节心血管系统、抗胃损伤、抑制前列腺素、升高外周血液白细胞的作用。
肉苁蓉为列当科植物肉苁蓉Cistanche deserticola Y.C.Ma的干燥带鳞叶的肉质茎。味甘、咸,性温。归肾、大肠经。功效为补肾阳,益精血,润肠通便。现代研究表明,肉苁蓉的主要成分是苯乙醇苷、环烯醚萜、木脂素和多糖类,肉苁蓉总苷、苯乙醇苷类和多糖类具有改善微循环,增加心、脑及外周末梢血管循环的作用,能够明显的保护心脑血管和神经系统。此外,肉苁蓉还有抗疲劳、抗衰老、提高免疫力、调节循环系统等作用,有利于患者身体达到健康平衡状态。
菟丝子为旋花科植物菟丝子Cuscuta chinensis Lam.的干燥成熟种子。味辛、甘,性平。归肝、肾、脾经。具有补益肝肾,固精缩尿,安胎,明目,止泻之功效。常用于肝肾不足,腰膝酸软,阳痿遗精,遗尿尿频,肾虚胎漏,胎动不安,目昏耳鸣,脾肾虚泻;外治白癜风等症。菟丝子含生物碱、蒽醌、香豆素、黄酮、甙类、甾醇、鞣酸、糖类等成分。具有壮阳、调节内分泌、调节血压、镇静、镇痛等作用。
《医方集解·补养之剂》指出“人之精与志,皆藏于肾,肾精不足则志气衰,不能上通于心,故迷惑善忘也。”故记忆力减退多由精血亏虚所引起,由于年老肝肾亏虚,精亏髓减,脑髓失所养或思虑过度劳伤心脾、情志不遂木郁克土、饮食不节伤及脾胃而致脾胃运化失司,气血生化乏源,不能上荣于脑,脑髓失所养,皆可致记忆力减退。治宜益精气,填精髓,补气血,宁心养神。本方以蚕蛹为君,性平味甘,益精气,健脾,生津止渴,营养先天之本;石菖蒲、远志芳香开窍,宁心安神,人参补益气血,强壮后天之本,三药共为臣药;五味子补肾宁心,肉苁蓉、益智仁、菟丝子补益肝肾,加强君药益精填髓之力,共为佐使药,全方共奏补精填髓之效。上述几种药材或食材通过合理的药物配伍,达到了改善睡眠的作用。
在其中一个实施例中,所述组合物的有效成分由以下重量份的原料制备而成:
蚕蛹10-15份,石菖蒲2-3份,远志2-3份,人参1-1.2份,五味子1-1.2份,益智仁1-1.2份,肉苁蓉1-1.2份,菟丝子1-1.2份。
在其中一个实施例中,所述组合物的有效成分由以下重量份的原料制备而成:
蚕蛹10份,石菖蒲2份,远志2份,人参1份,五味子1份,益智仁1份,肉苁蓉1份,菟丝子1份。
本发明还公开一种上述的用于增强学习记忆力的组合物的制备方法,包括以下步骤:
制备浸膏一:按重量份称取蚕蛹,粉碎,得粗粉,按蚕蛹原料重量的10-15倍加水浸泡,随后以盐酸调节溶液pH值为1.5-2.5,再按蚕蛹重量百分比的0.3-0.6%加入胃蛋白酶和按蚕蛹重量百分比的0.3-0.6%加入胰蛋白酶,37℃酶解3-6小时,然后加热使酶失活,过滤,得酶解液,将酶解液浓缩,即得浸膏一;
制备浸膏二:按重量份称取石菖蒲、远志和人参,按石菖蒲、远志和人参总重量的6-10倍加入体积百分含量为60%-80%乙醇溶液,回流提取2-4次,每次回流提取时间为2-4小时,合并滤液,将滤液浓缩,即得浸膏二;
制备浸膏三:按重量份称取五味子、益智仁、肉苁蓉和菟丝子,按五味子、益智仁、肉苁蓉和菟丝子总重量的10-15倍加入水,在80-100℃下浸提2-4次,每次浸提时间为2-4小时,合并滤液,将滤液浓缩,即得浸膏三;
混合:将上述得到的浸膏一、浸膏二和浸膏三混合均匀,即得具有增强学习记忆力功能的组合物。该组合物再经真空干燥,粉碎,得到粉末状的浸膏,命名为组合物1。
本发明的制备方法,采用现代制药技术提取原料中的有效成份,在保证疗效的前提下,提高了便利性和口感。并且在制备蚕蛹浸膏一时,以胃蛋白酶和胰蛋白酶进行酶解,使蚕蛹蛋白质中的部分肽键水解,获得吸收速度更快、生物活性更强的小分子多肽,使其更利于人体吸收,增加蚕蛹的生物利用度。
在其中一个实施例中,制备浸膏一步骤中,所述胃蛋白酶和胰蛋白酶的重量比为2:1-1:2。
在其中一个实施例中,制备浸膏一步骤中,将酶解液以截留分子量为50000的超滤柱进行过滤,得滤液,滤液减压浓缩,即得浸膏一。去除大分子杂质,保留蚕蛹酶解所得的多肽、腺苷、多糖、多种维生素等主要活性成份,对蚕蛹的有效成份进行纯化。
在其中一个实施例中,制备浸膏一步骤中,加热至100℃保持20-30分钟使酶失活。
在其中一个实施例中,制备浸膏一步骤中,滤液减压浓缩至55-65℃时密度为1.15-1.25g/mL的稠浸膏,再经真空干燥,粉碎,得到粉末状的浸膏一;
制备浸膏二步骤中,滤液减压浓缩至55-65℃时密度为1.15-1.25g/mL的稠浸膏,再经真空干燥,粉碎,得到粉末状的浸膏二;
制备浸膏三步骤中,滤液减压浓缩至55-65℃时密度为1.15-1.25g/mL的稠浸膏,再经真空干燥,粉碎,得到粉末状的浸膏三。
本发明还公开了一种上述的组合物在制备用于增强学习记忆力的药物或保健食品中的应用。
在其中一个实施例中,所述药物或保健食品的剂型为片剂、颗粒剂、硬胶囊、软胶囊、口服液、丸剂或滴丸。
与现有技术相比,本发明具有以下有益效果:
本发明的一种用于增强学习记忆力的组合物,原料由蚕蛹、石菖蒲、远志、人参、五味子、益智仁、肉苁蓉和菟丝子组成。这几种药材和食材相互配合,协同作用,具有良好的增强学习记忆力作用,同时还是抗衰老抗氧化和增强免疫力的代表中药,尤其适用于老年学习记忆力减退患者。并且,这些原料均为食材或药食两用的药材,符合中医“药食同源”理论,天然、毒副作用低,适合长期服用。
对比例1
石菖蒲、远志、人参组合物,通过以下原料制备而成:
石菖蒲120克,远志120克,人参60克。
具体制备方法如下:以上三味,按原料总重量的10倍量加入体积百分含量为70%的乙醇溶液,回流提取3小时,过滤,滤渣再以同样的条件提取2次,合并滤液,在0.06MPa的压力下以60℃浓缩至密度为1.15-1.25g/mL(60℃测)的稠浸膏;再在0.06 MPa的压力下以65℃干燥成干浸膏;干浸膏经粉碎,过80目筛,得粉末状的石菖蒲、远志、人参组合物。命名为组合物2。
对比例2
远志、人参、肉苁蓉、菟丝子组合物,通过以下原料制备而成:
远志120克,人参60克,肉苁蓉60克、菟丝子60克。
具体制备方法如下:按配方比称取远志和人参,按原料总重量的10倍量加入体积百分含量为70%的乙醇溶液,回流提取3小时,过滤,滤渣再以同样的条件提取2次,合并滤液,在0.06MPa的压力下以60℃浓缩至密度为1.15-1.25g/mL(60℃测)的远志、人参浸膏;
按配方比称取肉苁蓉和菟丝子,按原料总重量的15倍量加入水,煎煮3小时,过滤,滤渣再以同样的条件提取2次,合并滤液,在0.06MPa的压力下以60℃浓缩至密度为1.15-1.25g/mL(60℃测)的肉苁蓉、菟丝子浸膏;
将上述两种浸膏混合,再在0.06 MPa的压力下以65℃干燥成干浸膏;干浸膏经粉碎,过80目筛,得粉末状的远志、人参、肉苁蓉、菟丝子组合物。命名为组合物3。
对比例3
石菖蒲、远志、五味子、益智仁、肉苁蓉、菟丝子组合物,通过以下原料制备而成:
石菖蒲120克、远志120克、五味子60克、益智仁60克、肉苁蓉60克、菟丝子60克。
具体制备方法如下:按配方比称取石菖蒲和远志,按原料总重量的10倍量加入体积百分含量为70%的乙醇溶液,回流提取3小时,过滤,滤渣再以同样的条件提取2次,合并滤液,在0.06MPa的压力下以60℃浓缩至密度为1.15-1.25g/mL(60℃测)的石菖蒲、远志浸膏;
按配方比称取五味子、益智仁、肉苁蓉和菟丝子,按原料总重量的15倍量加入水,煎煮3小时,过滤,滤渣再以同样的条件提取2次,合并滤液,在0.06MPa的压力下以60℃浓缩至密度为1.15-1.25g/mL(60℃测)的五味子、益智仁、肉苁蓉、菟丝子浸膏;
将上述两种浸膏混合,再在0.06 MPa的压力下以65℃干燥成干浸膏;干浸膏经粉碎,过80目筛,得粉末状的石菖蒲、远志、五味子、益智仁、肉苁蓉、菟丝子组合物。命名为组合物4。
对比例4
石菖蒲、远志、人参、五味子、益智仁、菟丝子组合物,通过以下原料制备而成:
石菖蒲120克、远志120克、人参60克、五味子60克、益智仁60克、菟丝子60克。
具体制备方法如下:按配方比称取石菖蒲、远志和人参,按原料总重量的10倍量加入体积百分含量为70%的乙醇溶液,回流提取3小时,过滤,滤渣再以同样的条件提取2次,合并滤液,在0.06MPa的压力下以60℃浓缩至密度为1.15-1.25g/mL(60℃测)的石菖蒲、远志、人参浸膏;
按配方比称取五味子、益智仁和菟丝子,按原料总重量的15倍量加入水,煎煮3小时,过滤,滤渣再以同样的条件提取2次,合并滤液,在0.06MPa的压力下以60℃浓缩至密度为1.15-1.25g/mL(60℃测)的五味子、益智仁、菟丝子浸膏;
将上述两种浸膏混合,再在0.06 MPa的压力下以65℃干燥成干浸膏;干浸膏经粉碎,过80目筛,得粉末状的石菖蒲、远志、人参、五味子、益智仁、菟丝子组合物。命名为组合物5。
对比例5
石菖蒲、远志、人参、五味子、益智仁、肉苁蓉、菟丝子组合物,通过以下原料制备而成:
石菖蒲120克、远志120克、人参60克、五味子60克、益智仁60克、肉苁蓉60克、菟丝子60克。
具体制备方法如下:按配方比称取石菖蒲、远志和人参,按原料总重量的10倍量加入体积百分含量为70%的乙醇溶液,回流提取3小时,过滤,滤渣再以同样的条件提取2次,合并滤液,在0.06MPa的压力下以60℃浓缩至密度为1.15-1.25g/mL(60℃测)的石菖蒲、远志、人参浸膏;
按配方比称取五味子、益智仁、肉苁蓉和菟丝子,按原料总重量的15倍量加入水,煎煮3小时,过滤,滤渣再以同样的条件提取2次,合并滤液,在0.06MPa的压力下以60℃浓缩至密度为1.15-1.25g/mL(60℃测)的五味子、益智仁、菟丝子浸膏;
将上述两种浸膏混合,再在0.06 MPa的压力下以65℃干燥成干浸膏;干浸膏经粉碎,过80目筛,得粉末状的石菖蒲、远志、人参、五味子、益智仁、肉苁蓉、菟丝子组合物。命名为组合物6。
实验例1 对Morris水迷宫行为的影响
将上述实施例和对比例制备得到的组合物粉末进行Morris水迷宫实验。
一、材料
1. 组合物,通过上述实施例和对比例制备得到的组合物1-6;
2. 注射用戊巴比妥钠,上海新亚药业有限公司;
3. 成年昆明小鼠,体重18-22g,雌性,购自军事医学科学院实验动物中心。
二、方法
1. 动物分组和处理
将昆明小鼠随机分为8组: 每组18只, 分别为空白对照组、记忆障碍模型组、组合物1-6组(2.0g· kg-1·d-1)。对照组和模型组小鼠灌胃给予生理盐水(20 mL·kg-1·d-1),组合物1-6组分别灌服2.0g· kg-1·d-1的组合物1-6,连续灌胃14 d,第15d除对照组外,其余各组均腹腔注射戊巴比妥钠(20 mg·kg-1)1次,建立记忆障碍动物模型。
2. Morris水迷宫行为测试
于造模结束后进行Morris水迷宫系统的学习记忆能力测试,历时5d,每天训练4次,每次分别从4个不同的象限将小鼠面向池壁放入水中,观察并记录小鼠在有效区的停留时间及穿越平台的次数。如果小鼠在60s内未找到平台,需将其引至平台,此时潜伏期记为60s,每次训练间隔60s。前4d为训练适应期,第5天记录小鼠寻找平台的运动轨迹,包括有效区停留时间、穿越平台的次数、到达平台的潜伏期,作为行为学实验的测试结果。
三、结果
实验结果如下表所示。
表1 各组小鼠Morris水迷宫试验潜伏期结果(n=18)
VS 对照组:*p<0.05,**p<0.01;VS模型组:#p<0.05,##p<0.01。
结果显示,与正常对照组相比,模型组小鼠有效区停留时间、穿越平台次数均缩短,到达平台的潜伏期延长(P<0.05,P<0.01),表明记忆障碍动物模型建模成功;
与模型组相比,组合物1组可显著延长小鼠有效区停留时间、增加穿越平台的次数,缩短到达平台的潜伏期(P<0.05,P<0.01),说明组合物1有增强小鼠学习记忆能力的作用;
与模型组相比,组合物2可延长小鼠有效区停留时间、增加穿越平台的次数(P<0.05),但效果没有组合物1明显。此外,组合物2虽然可以缩短实验小鼠到达平台的潜伏期时间,但无统计学意义;
与模型组相比,组合物3可增加小鼠穿越平台的次数、缩短到达平台的潜伏期(P<0.05),但效果没有组合物1明显。此外,组合物3虽然可以延长实验小鼠有效区停留时间,但无统计学意义;
与模型组相比,组合物4可延长小鼠有效区停留时间、增加穿越平台的次数(P<0.05),但效果没有组合物1明显。此外,组合物4虽然可以缩短小鼠到达平台的潜伏期,但无统计学意义;
与模型组相比,组合物5可增加穿越平台的次数,缩短到达平台的潜伏期(P<0.05),但效果没有组合物1明显。此外,组合物5虽然可以延长实验小鼠有效区停留时间,但无统计学意义;
与模型组相比,组合物6可延长小鼠有效区停留时间、增加穿越平台的次数(P<0.05),但效果没有组合物1明显。此外,组合物6虽然可以缩短小鼠到达平台的潜伏期,但无统计学意义。
实验例2 对氧化应激相关酶活性及含量的影响
将上述实施例和对比例制备得到的组合物粉末进行氧化应激相关酶活性及含量测定实验。
一、材料
1. 组合物,通过上述实施例和对比例制备得到的组合物1-6;
2. 超氧化物歧化酶(superoxide dismutase,SOD)试剂盒,购自南京建成公司;
3. 一氧化氮(nitricoxide,NO)试剂盒,购自碧云天生物技术公司;
4. 过氧化氢酶(catalase,CAT)试剂盒,购自南京建成公司;
5. 实验动物同实验例1。
二、方法
实验例1中动物行为学实验结束后,取小鼠脑组织,用生理盐水制备10%的脑组织匀浆,4℃,4000 r·min-1离心15min,取上清液,按照试剂盒要求分别采用黄嘌呤氧化酶法、化学发光法、分光光度法测定脑组织SOD,NO,CAT 的活性。
三、结果
实验结果如下表所示。
表2 组合物对学习记忆障碍小鼠脑组织氧化应激的酶的影响(n=18)
组别 | 剂量/ g·kg-1 | CAT/U·mg(prot)-1 | SOD/U·mg(prot)-1 | NO/μmol·L-1 |
对照组 | 23.52±6.36 | 275.52±65.61 | 6.54±3.66 | |
模型组 | 10.36±2.28** | 154.05±26.89** | 32.65±9.91** | |
组合物1 | 2.0 | 26.82±6.75## | 260.36±59.21## | 10.74±4.22## |
组合物2 | 2.0 | 20.91±7.31# | 204.25±35.32# | 21.75±1.73 |
组合物3 | 2.0 | 13.93±3.35 | 201.18±42.46# | 18.06±5.94# |
组合物4 | 2.0 | 19.58±5.22# | 182.71±32.63 | 16.35±4.27# |
组合物5 | 2.0 | 15.62±4.81 | 213.54±55.69# | 16.88±6.31# |
组合物6 | 2.0 | 20.06±6.35# | 194.36±44.92# | 23.61±3.62 |
VS 对照组:*p<0.05,**p<0.01:VS模型组:#p<0.05,##p<0.01。
结果显示,模型组小鼠脑内CAT、SOD活性低于正常对照组(P<0.01),NO含量高于对照组(P <0.01),表明造模成功;
与模型组相比,组合物1组可显著提高小鼠脑内CAT的活性,显著提高小鼠脑组织内的SOD活性并显著降低NO含量(P<0.01);
与模型组相比,组合物2组可提高小鼠脑内CAT的活性,提高小鼠脑组织内的SOD活性(P<0.05),但效果没有组合物1明显。此外,组合物2虽然可以降低小鼠脑组织内NO含量,但无统计学意义;
与模型组相比,组合物3组可提高小鼠脑组织内的SOD活性,降低小鼠脑组织内NO含量(P<0.05),但效果没有组合物1明显。此外,组合物3虽然可以提高小鼠脑内CAT的活性,但无统计学意义;
与模型组相比,组合物4组可提高小鼠脑组织内的CAT活性,降低小鼠脑组织内NO含量(P<0.05),但效果没有组合物1明显。此外,组合物3虽然可以提高小鼠脑内SOD的活性,但无统计学意义;
与模型组相比,组合物5组可提高小鼠脑组织内的SOD活性,降低小鼠脑组织内NO含量(P<0.05),但效果没有组合物1明显。此外,组合物5虽然可以提高小鼠脑内CAT的活性,但无统计学意义;
与模型组相比,组合物6组可提高小鼠脑内CAT的活性,提高小鼠脑组织内的SOD活性(P<0.05),但效果没有组合物1明显。此外,组合物6虽然可以降低小鼠脑组织内NO含量,但无统计学意义。
实验例3 对小鼠脑组织中单胺递质含量的影响
将上述实施例和对比例制备得到的组合物粉末进行小鼠脑组织中单胺递质含量测定实验。
一、材料
1. 组合物,通过上述实施例和对比例制备得到的组合物1-6;
2. 去甲肾上腺素(norepinephrine,NE)、多巴胺盐酸盐(dopaminehydrochloride,DA)、5-羟色胺盐酸盐(5-hydroxytryptamine hydrochloride,5-HT)均购自美国Sigma公司;
3. 甲醇(色谱纯)、柠檬酸三钠购自Merck公司;
4. 水为去离子水;
5. 实验动物同实验例1。
二、方法
在Morns水迷宫实验结束之后,每组取半数小鼠,用于检测脑组织中单胺递质的含量。将小鼠断头,冰上取脑组织400mg,加0.1 mol/L高氯酸(2ml),冰浴匀浆, 4℃,14000 r·min-1离心20min,取上清,0.45μm滤膜(Millipore,USA)过滤,注入高效液相色谱仪,进样100μL。
流动相:柠檬酸三钠(20mmol/L),盐酸调pH4.50,0.45μm滤膜过滤,加5%(V/V)甲醇,超声脱气,流速:1.0ml/min,柱温:5℃,荧光检测器波长:激发波280nm,发射波315nm;峰面积定量。
三、结果
实验结果如下表所示。
表3 组合物对学习记忆障碍小鼠脑组织中NE,DA,5-HT 含量的影响(n=18)
组别 | 剂量/ g·kg-1 | NE/ng·g-1 | DA/ng·g-1 | 5-HT/ng·g-1 |
对照组 | 5.54±1.32 | 1.25±0.41 | 1.72±0.36 | |
模型组 | 7.82±1.21* | 1.52±0.35* | 0.65±0.39* | |
组合物1 | 2.0 | 4.34±0.92## | 1.16±0.22# | 1.82±0.48## |
组合物2 | 2.0 | 5.22±1.57# | 1.39±0.26 | 1.38±0.45# |
组合物3 | 2.0 | 6.26±0.96 | 1.35±0.24 | 1.29±0.32# |
组合物4 | 2.0 | 5.45±1.26# | 1.31±0.32 | 1.15±0.41# |
组合物5 | 2.0 | 6.07±1.13 | 1.22±0.29# | 1.24±0.27# |
组合物6 | 2.0 | 5.66±1.35# | 1.26±0.38# | 0.92±0.32 |
VS 对照组:*p<0.05,**p<0.01;VS模型组:#p<0.05,##p<0.01。
结果显示,与对照组比较,模型组小鼠脑组织中NE,DA含量增加,5-HT含量降低,含量变化均具有统计学意义(P<0.05),表明建模成功;
与模型组比较,组合物1组小鼠脑组织中NE,DA含量明显降低,5-HT含量明显升高,具有统计学意义(P<0.05, P<0.01);
与模型组比较,组合物2组小鼠脑组织中NE含量降低,5-HT含量升高,具有统计学意义(P<0.05),但效果没有组合物1明显。此外,组合物2虽然可以降低小鼠脑组织中DA含量,但无统计学意义;
与模型组比较,组合物3组小鼠脑组织中5-HT含量升高,具有统计学意义(P<0.05),但效果没有组合物1明显。此外,组合物3虽然可以降低小鼠脑组织中NE和DA含量,但无统计学意义;
与模型组比较,组合物4组小鼠脑组织中NE含量降低,5-HT含量升高,具有统计学意义(P<0.05),但效果没有组合物1明显。此外,组合物4虽然可以降低小鼠脑组织中DA含量,但无统计学意义;
与模型组比较,组合物5组小鼠脑组织中DA含量降低,5-HT含量升高,具有统计学意义(P<0.05),但效果没有组合物1明显。此外,组合物5虽然可以降低小鼠脑组织中NE含量,但无统计学意义;
与模型组比较,组合物6组小鼠脑组织中NE,DA含量降低,具有统计学意义(P<0.05),但效果没有组合物1明显。此外,组合物6虽然可以升高小鼠脑组织中5-HT含量,但无统计学意义。
Claims (9)
1.一种用于增强记忆力的组合物,其特征在于:所述组合物的有效成分由以下重量份的原料制备而成:蚕蛹10-30份,石菖蒲2-10份,远志2-10份,人参1-5份,五味子1-5份,益智仁1-5份,肉苁蓉1-5份,菟丝子1-5份。
2.一种用于增强记忆力的组合物,其特征在于:所述组合物的有效成分由以下重量份的原料制备而成:蚕蛹10-15份,石菖蒲2-3份,远志2-3份,人参1-1.2份,五味子1-1.2份,益智仁1-1.2份,肉苁蓉1-1.2份,菟丝子1-1.2份。
3.一种用于增强记忆力的组合物,其特征在于:所述组合物的有效成分由以下重量份的原料制备而成:蚕蛹10份,石菖蒲2份,远志2份,人参1份,五味子1份,益智仁1份,肉苁蓉1份,菟丝子1份。
4.一种如权利要求1-3中任一项所述用于增强记忆力的组合物的制备方法,其特征在于:包括以下步骤:
制备浸膏一:按重量份称取蚕蛹,粉碎,得粗粉,按蚕蛹原料重量的10-15倍加水浸泡,随后以盐酸调节溶液pH值为1.5-2.5,再按蚕蛹重量百分比的0.3-0.6%加入胃蛋白酶和按蚕蛹重量百分比的0.3-0.6%加入胰蛋白酶,37℃酶解3-6小时,然后加热使酶失活,过滤,得酶解液,将酶解液浓缩,即得浸膏一;
制备浸膏二:按重量份称取石菖蒲、远志和人参,按石菖蒲、远志和人参总重量的6-10倍加入体积百分含量为60%-80%乙醇溶液,回流提取2-4次,每次回流提取时间为2-4小时,合并滤液,将滤液浓缩,即得浸膏二;
制备浸膏三:按重量份称取五味子、益智仁、肉苁蓉和菟丝子,按五味子、益智仁、肉苁蓉和菟丝子总重量的10-15倍加入水,在80-100℃下浸提2-4次,每次浸提时间为2-4小时,合并滤液,将滤液浓缩,即得浸膏三;
混合:将上述得到的浸膏一、浸膏二和浸膏三混合均匀,再经真空干燥,粉碎,得到粉末状的浸膏,即可制得为所述组合物。
5.根据权利要求4所述的制备方法,其特征在于:在制备蚕蛹浸膏一时,以胃蛋白酶和胰蛋白酶进行酶解,使蚕蛹蛋白质中的部分肽键水解;制备浸膏一步骤中,所述胃蛋白酶和胰蛋白酶的重量比为2:1-1:2;制备浸膏一步骤中,将酶解液以截留分子量为50000的超滤柱进行过滤,得滤液,滤液减压浓缩;制备浸膏一步骤中,加热至100℃保持20-30分钟使酶失活;制备浸膏一步骤中,滤液减压浓缩至55-65℃时密度为1.15-1.25g/mL的稠浸膏,再经真空干燥,粉碎,得到粉末状的浸膏一。
6.根据权利要求5所述的制备方法,其特征在于:制备浸膏二步骤中,滤液减压浓缩至55-65℃时密度为1.15-1.25g/mL的稠浸膏,再经真空干燥,粉碎,得到粉末状的浸膏二。
7.根据权利要求6所述的制备方法,其特征在于:制备浸膏三步骤中,滤液减压浓缩至55-65℃时密度为1.15-1.25g/mL的稠浸膏,再经真空干燥,粉粹,得到粉末状的浸膏三。
8.一种如权利要求1-3中任一项所述用于增强记忆力的组合物在制备用于增强记忆力的保健食品中的应用。
9.如权利要求8所述保健食品的剂型为片剂、颗粒剂、硬胶囊、软胶囊、口服液、丸剂或滴丸。
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